Identification, Molecular Characterization, and Chromosomal Localization of the cDNA Encoding a Novel Leucine Zipper Motif-Containing Protein

GENOMICS

36, 54 –62 (1996) 0425

ARTICLE NO.

Identification, Molecular Characterization, and Chromosomal Localization of the cDNA Encoding a Novel Leucine Zipper Motif-Containing Protein DER-SHAN SUN,* ALICE C. CHANG,†,‡ NANCY A. JENKINS,§ DEBRA J. GILBERT,§ NEAL G. COPELAND,§ AND NAN-CHI A. CHANG*,‡,1 *Institute of Microbiology & Immunology and †Institute of Neuroscience, ‡Center for Neuroscience, National Yang-Ming University, Taipei, Taiwan 112, Republic of China; and §Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received January 16, 1996; accepted May 9, 1996

tagenesis studies later provided unequivocal evidence supporting the notion that leucine zipper motifs are required for homo- and/or heterodimerization of Fos and Jun, which then leads to proper juxtaposition of an adjacent basic domain present in both proteins prior to sequence-specific DNA binding (Gentz et al., 1989; Turner and Tjian, 1989; Busch and Sassone-Corsi, 1990). Leucine zipper motifs are also present in other types of proteins lacking the basic motif. They have been implicated as negative transcription regulators by forming a nonfunctional heterodimeric complex with those transcriptional factors containing the basic domains (Kageyama and Pastan, 1989; Ron and Habener, 1992; Bange et al., 1994). Similar negative regulators have also been reported for bHLH transcription factors (Benezra et al., 1990). More recently, leucine zipper motifs were found in the membrane-bound glucose transporter (Asano et al., 1992), voltage-gated potassium channel (Sokol et al., 1994), cytoplasmic protein kinase (Reddy and Pleasure, 1994), and nuclear envelope anchoring protein Nup 107 (Radu et al., 1994). A basic region extending beyond the leucine repeats toward the amino terminus was not found in any of these proteins. During the course of cloning an immune responserelated gene from a mouse bone marrow cDNA library, we identified a partial cDNA clone K encoding a novel protein with two leucine zipper motifs at its amino terminus. We have tentatively designated this protein LUZP. Considering the aforementioned roles for leucine zipper-containing proteins known to date, exploration of the physiological significance of this distinct LUZP would be of fundamental importance. We have since completed the molecular cloning of its cDNA from libraries constructed from mouse activated peritoneal exudate cells (PEC) and examined its tissue distribution. We have also determined the chromosomal location of Luzp in mouse to determine whether Luzp maps near any mouse

cDNA clones encoding a novel protein (LUZP) with three leucine zipper motifs were first identified from a murine bone marrow cDNA library. After screening two additional cDNA libraries of activated peritoneal exudate cells, 32 positive clones were obtained from 1.3 1 10 7 phage plaques. Four overlapping clones constituting a total of 7399 bp were sequenced on both strands. The complete open reading frame of LUZP is 1067 amino acids. In addition to three leucine zipper motifs located at the NH2 terminus, there are three nuclear localization signals and a large number of putative Ser/Thr phosphorylation sites. Western blot analyses indicate that LUZP is predominantly expressed in brain, whereas immunocytochemistry data clearly reveal its presence in the nucleus of neurons. Interspecific backcross analyses have mapped Luzp to mouse chromosome 4 in proximity to Gpcr14. Comparative mapping data suggest that the human homolog of Luzp will map to human chromosome 1p36. q 1996 Academic Press, Inc.

INTRODUCTION

The leucine zipper motif was first described as a salient structural feature common to transcription factors such as C/EBP, GCN4, and oncogene products Myc, Fos, and Jun. The motif is characterized by the presence of a heptad periodicity of leucine residues spanning some 28 to 35 amino acids. When projecting the sequence on an idealized a-helical wheel, the leucine repeat was even more conspicuous, lending a proposed role in protein dimerization (Landschulz et al., 1988; Johnson and Mcknight, 1989; Struhl, 1989). MuSequence data from this article have been deposited with the GenBank/EMBL Data Libraries under Accession No. L49344. The gene symbol Luzp was approved by the mouse nomenclature committee MGD. 1 To whom correspondence should be addressed. Telephone: 02826-7114. Fax: 02-820-0259. 0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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mutations that are good candidates for a mutation in this gene. MATERIALS AND METHODS Library screening and DNA sequencing. A partial cDNA clone (K, 550 bp) of Luzp, derived from a mouse bone marrow cDNA library (DBA/2, Clontech), was used as a probe in initial screenings of two cDNA libraries ( lgt11 and LambdaZAPII) customconstructed from activated PEC of ICR mice by Clontech. Subsequently, K clone and/or various walking probes were used for amplification and purification of overlapping clones of Luzp. Inserts of positive clones obtained from lgt11 were amplified by polymerase chain reaction and sublconed into pBluescript II vectors prior to sequence determination. Those obtained from LambdaZAPII were just excised by helper phage (Short et al., 1988). Sequence of both strands were determined by the dideoxy chain termination method (Sanger et al., 1977) using the Sequenase DNA sequencing kit (United States Biochemical). Fusion protein and antibody production. A DNA fragment (425 bp) from K clone containing the first two leucine zipper motifs was cloned into pGEMEX prokaryotic expression vector (Promega) and transformed into bacteria strain JM109 (DE3). Transformants were induced to express fusion protein containing of T7 gene 10 and leucine zipper motif-containing peptide of LUZP by IPTG (1 mM). The fusion protein was excised from 10% SDS– PAGE, homogenized with incomplete Freund’s adjuvant, and injected into rabbits for antibody production. The specificity and titer of the antibodies obtained were characterized by Western blot. A second polyclonal antibody was raised the same way against fusion protein composed of another fragment (296 bp) of Luzp expressed in a pRSET-A system (Invitrogen). To ensure that antibodies 425 and 296 recognize the same protein, radioimmunoprecipitation results obtained by employing the two antibodies were compared. In vitro transcription and translation of a rat brain cDNA clone R51R containing the entire coding region of LUZP was carried out using the TNT coupled reticulocyte lysate system (Promega). [ 35S]methionine-labeled translational product was immunoprecipitated with 425 or 296, collected by milk-preabsorbed protein A–Sepharose (Pharmacia), washed, and fractionated on 7.5% SDS– PAGE before autoradiography. Preimmune serum of respective rabbits was used as the negative control. Western blot analyses and immunocytochemistry. Various tissues (0.1 g) were homogenized in 5 ml of solution containing 20 mg/ml DNase, RNase, 1.4 mg/ml pepstatin, 1 mg/ml leupeptin, and 80 mg/ml phenylmethylsulfonyl fluoride using a tissue tearor (Biospec Products, Model 985-370). The protein extracts were aliquoted and diluted 1:1 with 21 SDS sample buffer. The protein concentration was determined by Coomassie blue protein microassay (Pierce). After denaturation by heat for 5 min, the proteins (150 mg/lane) were fractionated on 6% SDS – PAGE (Laemmli, 1970) and electroblotted onto Immobilon-PVDF membrane (Millipore) for immunodetection using antibody 425 (1:500 dilution) according to Towbin and Gordon (1984) with minor modifications. Immunocytochemical staining was carried out on vibratome sections of rat brain after being perfusion fixed with 4% paraformaldehyde in 0.12 M PB (pH 7.4) according to the protocol of Sternberger et al. (1970). For immunodetection of both Western blots and brain sections, prior to incubation with any antibodies, the membranes were blocked with 5% delipided milk (or BSA) in TBS2 (50 mM Tris – HCl, pH 7.5, 150 mM NaCl) for 30 min to minimize nonspecific binding. The secondary goat-anti-rabbit IgG antibody and the rabbit peroxidase anti-peroxidase were obtained from Jackson ImmunoResearch Lab. Inc. The positive reaction products were developed in TBS-2 containing 0.05% H 2O 2 and 0.05% 3 *, 3-diaminobenzidine-tetrahydrochloride (Sigma). Interspecific backcross mapping. Interspecific backcross progeny were generated by mating (C57BL/6J 1 Mus spretus) F1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 backcross mice were used to map the Luzp

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locus (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (Jenkins et al., 1982). All blots were prepared with Hybond-N / nylon membrane (Amersham). The probe, a 987-bp fragment of mouse cDNA (clone p22), was labeled with [ a -32P]dCTP using a random prime labeling kit (Stratagene); washing was performed to a final stringency of 1.01 SSCP, 0.1% SDS, 657C. Two fragments of 4.2 and 1.8 kb were detected in HindIII-digested C57BL/6J DNA, while 4.3- and 1.9-kb fragments were detected in M. spretus DNA. In addition, an 11.5-kb fragment was detected in SphI-digested DNA, while a 20.5-kb fragment was detected in M. spretus DNA. The presence or absence of the 1.9-kb M. spretus-specific HindIII fragment and the 20.5-kb M. spretus-specific SphI fragment was followed in backcross mice. The two sets of data were combined when determining the map position of Luzp. A description of the probes and restriction fragment length polymorphisms (RFLPs) for the loci linked to Luzp, including Gardner-Rasheed feline sarcoma viral oncogene homolog (Fgr), G-protein-coupled receptor 14 (Gpcr14), and natriuretic peptide precursor type A (Nppa), have been reported previously (Ceci et al., 1989, Wilkie et al., 1993). Recombination distances were calculated as described by Green (1981) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of double and multiple recombination events across the chromosome.

RESULTS AND DISCUSSION

cDNA Cloning and DNA Sequence Analyses We have obtained a cDNA clone K while screening an immune response-related gene (Ym1) (Chang et al., unpublished data) with its specific polyclonal antibody from an expression library of mouse bone marrow. DNA sequence data later indicated that clone K does not encode YM1 but rather a novel protein containing two leucine zipper motifs. This protein has since been designated LUZP. To obtain the full-length cDNA of Luzp, clone K was used as a probe to rescreen the murine (DBA/2) bone marrow cDNA library, and no further cDNA clones were identified. Due to the apparent low message abundance of Luzp in bone marrow, we subsequently screened two additional libraries constructed from activated peritoneal exudate cells harvested from ICR mice. After six rounds of amplification and purification with walking probes, 32 positive clones were obtained from 1.3 1 107 clones. Four clones, P13, P22, P34, and AC45, were subcloned into pBluescript II vector for sequence determination of both strands. The composite length of these four overlapping cDNA clones is 7399 bp, containing an open reading frame from nucleotides 259 to 3459 capable of encoding a protein of 1067 amino acids, a relatively short 5*-untranslated region (5*UTR, 258 bp), and a long 3*-untranslated region (3*UTR, 3940 bp). A consensus polyadenylation signal AATAAA is located 3899 bp downstream from the stop codon and 16 bp upstream from the poly(A) tract (Fig. 1). Although the functional significance of a relatively long 3*-untranslated region is not clear at this point, recent reports have indicated that AU-rich motifs present in the 3 * UTR are responsible for rapid degradation of mRNAs of c-fos, c-myc, interleukin, lympho-

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FIG. 1. Nucleotide sequence and deduced amino acid sequence of LUZP. The initiation codon ATG is underlined. The stop codon TGA is marked by an asterisk. Leucine residues within the leucine zipper motifs are circled. The consensus sequences of NLS are boxed. The polyadenylation signal AATAAA is highlighted in boldface. The AU-rich motifs in the 3 *-untranslated region are underlined.

kines, GM-colony stimulating factors, and b2-adrenoceptor (Shaw and Kamen, 1986; Reeves et al., 1987; Shyu et al., 1991; Huang et al., 1993). There are also 14 AU-rich motifs in the 3*-untranslated region of Luzp. Whether these redundant AU-rich elements play any role in destabilizing Luzp mRNA will await further studies. A sequence homology search in the GenBank database (PC Gene, Intelligenetics) sug-

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gested that Luzp is a novel gene. Genomic Southern blot analyses indicate that only one copy of Luzp is present per haploid genome in mouse (data not shown). Zooblot analyses were also carried out using K clone as probe. Under relatively low-stringency conditions, Luzp was not detected in yeast, plants (corn and wheat), Drosophila, or lower vertebrates such as fish. Luzp is, however, unequivocally present in

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FIG. 1—Continued

higher vertebrates, i.e., rat, mouse, and human (data not shown). Characteristics of LUZP Amino Acid Sequence The amino acid sequence deduced from the cDNA revealed that LUZP is 1067 amino acids long, with a calculated molecular mass of 119 kDa and a theoretical isoelectric point of 8.34. By a search with the computer program PCGENE PROSITE, there are three leucine

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zipper motifs located at the NH2 terminus: residues 16–44, 106–127, and 158– 186 (Fig. 1). When projected onto the helical wheel structure, the charged amino acid residues are clustered on one side, whereas the periodic leucine and hydrophobic amino acid residues are located on the other side. All of the above characteristics fulfill the signature of leucine zipper motifs reported for other known proteins (e.g., Fos and Jun) (Johnson and Mcknight, 1989; Struhl, 1989). However, the consensus basic motif (about 30 amino acids) lo-

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FIG. 2. Alignment of the amino acid sequences of mouse and rat LUZP (mLuzp and rLuzp, respectively). A colon denotes that two aligned residues are identical, whereas a dot indicates that the two residues are similar. (j) denotes leucine residues in the zipper motifs. NLSs are boxed.

cated immediately NH2-terminal to the leucine zipper motif is absent in LUZP. Despite the absence of this basic motif, which has been implicated in DNA binding, the basic pI (8.34, by PC GENE) and the amino acid composition analyses revealed a high content of basic residues, i.e., Lys (9.2%) and Arg (7.4%). The protein is also rich in Ser (133 residues, 12.5%) and Thr (60 residues, 5.6%). We have therefore searched for putative phosphorylation sites; there are 30 casein kinase II sites, 30 protein kinase C sites, and two protein ki-

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nase A sites. There are in addition eight potential glycosylation sites and three sets of peptide sequence, i.e., KKMR317, KRER402, and KRGR469 (Fig. 1), corresponding to the consensus sequence of nuclear localization signals KR/K-X-R/K (Nath and Nayak, 1990; Meier and Blobel, 1992; Srinivasan et al., 1994). When compared to the homologous gene of rat (Sun et al., unpublished data), the two coding regions share as high as 91% homology (Fig. 2). There are also three leucine zipper motifs in the rat homolog at regions comparable to

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those in the murine counterpart. The same is true for three of four nuclear localization signals found in the rat homolog (Fig. 2). The only notable difference exists adjacent to the carboxy-terminal region, where a 6amino-acid (LKCSED) deletion is present in the murine homolog (Fig. 2). Tissue Distribution With a novel gene on hand whose protein product bears many intriguing biochemical characteristics, we proceeded with the mapping of its expression pattern in various tissues by Western blot and immunocytochemistry. Antibody 425 was so designated because it was prepared by expressing the DNA fragment (425 bp) encoding the first two leucine zipper motifs in the pGEMEX system (amino acid residues 1–124, Fig. 3A). Subsequently, the antibody was elicited in rabbits using SDS –PAGE-purified fusion protein. The specificity of the antibody was demonstrated in Western blot by recognizing the fusion protein in bacteria extracts after IPTG induction (Fig. 3A). The expression of LUZP was then examined in tissues, i.e., brain, kidney, thymus, liver, lung, spleen, lymph node, ovary, adrenal gland, bone marrow, and activated PEC. Using 150 mg protein extracted from each tissue and antibody 425 diluted 500- to 1000-fold, Western blot data clearly indicated that LUZP is predominantly expressed in brain with an estimated molecular weight of about 140 kDa (Fig. 3B). While kidney and lung express barely detectable LUZP of the same molecular weight, a cross-reactive protein band of significantly higher molecular weight was consistently detected in these tissues. Despite the fact that cDNA libraries of bone marrow and activated PEC were the original sources of Luzp cDNA clones, LUZP expression was apparently below detection sensitivity in the immunoblot system used in the present study (Fig. 3B). Antibody 425 was effective in Western blot analyses; nevertheless, it was not as effective when employed in immunocytochemistry. It is possible that in its native conformation within the sections, the epitopes of LUZP, those residing in the leucine zipper motifs-containing region, were not accessible. Elicited against another region of LUZP (amino acid residues 336 to 434), the specificity of antibody 296 was evaluated by examining whether it can recognize and precipitate the same translational product of Luzp as antibody 425. Radioimmunoprecipitation results as shown in Fig. 3C indicated that indeed antibodies 425 and 296 both recognize a translational product derived from a DNA template (R51R) containing the entire open reading frame of LUZP. Subsequently, antibody 296 has been evaluated as effective in immunocytochemistry. In the adult rat brain, LUZP is expressed in cerebral cortex, cerebellum, hippocampus, and brain stem. A representative micrograph of LUZP in the temporal lobe of cerebral cortex illustrates its subcellular localization. As revealed in Fig. 3D, LUZP immunoreactivity is present

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in both cell soma and dendritic profiles of neurons; however, predominant staining resides within its cell nucleus. This is consistent with the fact that there exist at least three motifs bearing consensus nuclear localization signals in the primary structure of LUZP (Fig. 1). The study of the temporal and spatial expression of LUZP in the central nervous system during development is currently under way. Chromosome Localization The mouse chromosomal location of Luzp was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J 1 M. spretus) F1 1 C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2000 loci that are well distributed among all mouse autosomes and the X chromosome (Copeland and Jenkins, 1991, unpublished results). C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative RFLPs using a mouse cDNA probe (clone P22) for Luzp (see Materials and Methods). M. spretus-specific HindIII and SphI fragments were used to follow the segregation of the Luzp locus in backcross DNAs. The mapping results indicated that Luzp is located in the distal region of mouse chromosome 4 (Fig. 4). Although 114 mice were analyzed for all four markers shown in the haplotype analysis (Fig. 4), up to 178 mice were analyzed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are centromere –Fgr –1/117–Luzp–0/178–Gpcr14–5/119–Nppa. The recombination frequencies (expressed as genetic distances in centimorgans { standard error) are centromere –Fgr –0.9 { 0.9–(Luzp, Gpcr14) –4.2 { 1.8– Nppa. The Luzp and Gpcr loci cosegregated in 178 animals typed in common, suggesting that the two loci are within 1.7 cM of each other at the 95% confidence limit. We have compared our interspecific linkage map of chromosome 4 with a composite map that reports the location of many uncloned mouse mutations (provided by GBASE, a computerized database of mouse linkage information maintained by The Jackson Laboratory, Bar Harbor, ME). This analysis indicated that one mutation, cribriform degeneration (cri), maps near Luzp. Homozygous cri mice are anemic, resulting from a deficiency of normocytic red cells, display an electrolyte imbalance, and show ataxic behavior beginning at 2.5 to 3 weeks of age (Green et al., 1972). Many homozygous cri mice die before weaning, and most are dead before 3 months of age. Given the expression of Luzp in brain, its respective cDNA clones identified originally from bone marrow, and activated PEC, Luzp should be considered as a candidate gene for cri. The distal half of mouse chromosome 4 shares a

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FIG. 4. Luzp maps in the distal region of mouse chromosome 4. Luzp was mapped to mouse chromosome 4 by interspecific backcross analysis. (Top) The segregation patterns of Luzp and flanking genes in 114 backcross animals typed in common are shown. For some individual pairs of loci, more than 114 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J 1 M. spretus) F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, while the white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. (Bottom) A partial chromosome 4 linkage map showing the location of Luzp in relation to linked genes is shown. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes are shown to the right. References for human map positions can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Medical Library of the Johns Hopkins University (Baltimore, MD).

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large region of homology with human chromosome 1 (Fig. 4). In particular, Luzp was localized between two loci, Fgr and Nppa, that have been mapped to human chromosome 1p36.2– p36.1 and 1p36, respectively. These comparative mapping results suggest the human homolog of Luzp will map to human 1p36 as well. Consistent with this hypothesis, Gpcr14 shows very strong homology to the human serotonin 1d receptor (HTR1D) (Wilkie et al., 1993), suggesting that Gpcr14 is the mouse homolog of this human receptor. The HTR1D receptor has been mapped to human chromosome 1p36.3–p34.3. In summary, from cDNA libraries of the immune system, we have identified a novel gene Luzp, which encodes a protein with three leucine zipper motifs at its amino terminus. The composite length of the corresponding cDNA reported in the present study is 7399 bp, containing an open reading frame capable of encoding a protein of 1067 amino acids. Molecular characterization of Luzp cDNA reveals, in addition to leucine zipper motifs, three nuclear localization signals and a large number of putative Ser/Thr phosphorylation sites. Western blot analyses indicate that LUZP is predominantly expressed in brain, whereas immunocytochemistry data have clearly revealed its presence in the nucleus of neurons. Interspecific backcross analysis has mapped Luzp to murine chromosome 4 in proximity to Gpcr14. Comparative mapping data suggest that the human homolog of Luzp will map to human 1p36. ACKNOWLEDGMENTS We thank Debbie Barnhart of NCI for excellent technical assistance and Dr. C. H. Lee, Department of Pathology, Indiana Universtiy Medical Center, Indianapolis for support in the initial stage of this study. This research was supported by grants (NSC-83-0412B010-054-M15 and NSC-84-2331-B010-098-M15 to NCC) provided by the National Science Council, ROC, and by the National Cancer Institute, DHHS, under Contract NO1-CO-46000 with ABL.

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FIG. 3. Tissue distribution and subcellular localization of LUZP. (A) Polyclonal antibody against LUZP was raised against pGEMEX expressed fragment 425. Western blot was carried out to verify specificity of the antibody. Lane 1, high-molecular-weight markers (Pharmacia); lane 2, JM109(DE3) extracts before IPTG induction; lane 3, after IPTG induction of fusion protein, lanes 1 through 3 were stained with Coomassie blue. The same samples as in lanes 2 and 3 were loaded in lanes 4 and 5, and 6 and 7 and immunoblotted with preimmune (4, 5) or immune (6, 7) serum, respectively. (B) Total proteins (150 mg per lane) extracted from different mouse tissues and activated PEC were separated on 6% SDS– PAGE. The blot was subsequently probed with antibody 425 (1:500 dilution). The arrow denotes the position of LUZP. (C) An additional polyclonal antibody against LUZP was elicited against fragment 296 of LUZP in the pRSET system. To verify that both antibodies 425 and 296 recognize the same LUZP, [35S]methionine-labeled translational product obtained using the TNT coupled reticulocyte lysate system from a rat cDNA clone containing the complete coding region of LUZP was subjected to immunoprecipitation analyses. As shown in the autoradiogram, both 425 and 296 (Im) precipitated the same protein as the labeled translational product (R51R) directly fractionated in the gel, whereas preimmune (Pre) serum of both were unable to precipitate the respective antigen. (D) Vibratome sections of the perirhinal cortex of rat brain were immunostained with antibody 296 (1:1000). LUZP-immunoreactive staining is present in nucleus and/or dendritic profiles of some neurons.

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gnma

AP: Genomics